Mechanisms Underlying H2O2-Evoked Carbonyl Modification of Cytoskeletal Protein and Axon Injury in PC-12 Cells

Abstract

Background/Aims: To investigate the mechanism that enables oxidative stress and cytoskeleton protein carbonylation to contribute to axonal dysfunction in traumatic brain injury (TBI). Methods: We created an in vitro model of neuronal oxidative damage by exposing a neuron-like cell line (PC-12) to different concentrations (100 μM, 200 μM, and 300 μM) of H₂O₂ for 24 h or 48 h. Carbonyl modification of cytoskeletal proteins (β-actin and β-tubulin) and its impact on β-actin/β-tubulin filament dynamics were determined by enzyme-linked immunosorbent assay, immunostaining, and western blotting. Depolymerization of β-actin/β-tubulin filaments was evaluated using the monomer/polymer ratio of each protein via western blotting. Phosphorylation of the neurofilament heavy chain (P-NFH) was used as an axonal injury marker and detected by immunostaining. Results: Our results showed that H₂O₂ treatment led to increased oxidative stress in PC-12 cells, as indicated by the increased generation of malondialdehyde and 8-hydroxydeoxyguanosine and decreased intracellular glutathione levels. H₂O₂ treatment also increased carbonyl modification of total proteins and cytoskeleton proteins β-actin/β-tubulin, which occurred concurrently with the suppression of proteasome activity. Moreover, H₂O₂ treatment increased the generation of the axonal injury marker P-NFH, and depolymerization of the β-actin/β-tubulin filaments was indicated by increased monomer/polymer ratios of each protein. Lastly, overexpression of the proteasome β5 subunit in PC-12 cells significantly reduced the H₂O₂-induced accumulation of carbonylated β-actin/ β-tubulin, P-NFH, and β-actin/β-tubulin depolymerization. Conclusions: We concluded that carbonylation of cytoskeleton proteins could lead to depolymerization of their filaments and axonal injury, and proteasome suppression contributes to the accumulation of carbonylated proteins under oxidative conditions.

Teriflunomide Preserves Neuronal Activity and Protects Mitochondria in Brain Slices Exposed to Oxidative Stress

“…Moreover, it has been reported that other factors, such as the depolymerization of cytoskeletal systems, may lead to impaired mitochondrial transportation. In this context, Zhang et al, 2018, demonstrated a H 2 O 2 -induced depolymerization of ß-actin filaments in neuronal cell lines incubated for 24 h with 200 µM H 2 O 2 [44]. Such an effect is, however, very improbable in our experimental brain slices, which were incubated for only 30 min with H 2 O 2 .…”

Carnosine synthase deficiency is compatible with normal skeletal muscle and olfactory function but causes reduced olfactory sensitivity in aging mice

“…Beside preventing carbonylation and other oxidative damage of proteins, carnosine may also inhibit cross-linking by reacting with carbonylated proteins (54). Carbonylation of tubulin can impair its polymerization and favor depolymerization of microtubules (55,56). Thus, it is possible that the increase of protein carbonylation in Carns1(-/-) mice may impair the functional properties of ORN axons in the OB, though further studies are necessary to solve this question.…”

Plasma Exosome-derived MicroRNAs as Novel Biomarkers of Traumatic Brain Injury in Rats

“…Many studies have indicated the role and the potential therapy of activation of RAS or RAP1 in traumatic brain injury [55][56][57]. There were also several studies about the actin cytoskeleton, which showed the mechanism in TBI procedure [58,59]. As the upstream pathway, MAPK signaling pathway may serve as another important role.…”